Pesquisa de genes de resistência do M. tuberculosis a isoniazida e rifampicina por sequenciamento e pela tecnologia de DNA-STRIP

Tuberculose (TB), causada por Mycobacterium tuberculosis, é uma das doenças infecciosas que mais causam mortes. Estima-se que mais de dois bilhões de pessoas estejam infectadas no mundo. O tratamento da TB consite em associação de fármacos, isoniazida, rifampicina, pirazinamida e etambutol, nos primeiros 2 meses e 4 meses de isoniazida e de rifampicina. Internacionalmente, são consideradas cepas multi resistentes (MDR), as que apresentam resistência simultânea a isoniazida e a rifampicina. A rápida detecção de resistência é essencial para o controle e tratamento da TB, reduzindo, assim, o custo do tratamento e a transmissão da doença. Neste projeto, os isolados já identificados fenotipicamente como resistentes a isoniazida e/ou rifampicina, foram submetidos ao sequenciamento de Sanger para pesquisa de 3 genes relacionados a resistência a isoniazida (katG, inhA e ahpC) e 1 gene de resistência a rifampicina (rpoB). Foi realizada uma comparação destes genes mutados para a resistência utilizando o novo teste desenvolvido pela Biomérieux, denominado GenoType® MTBDRplus, que se baseia na tecnologia DNA-STRIP. Através deste novo teste, foi observada mutação em 22 isolados clínicos de M. tuberculosis para genes de resistência a isoniazida e/ou rifampicina, sendo 4 provenientes do MS e 18 de SP. Já pelo sequenciamento genético foi observada mutação em 24 isolados para genes de resistência a isoniazida e/ou rifampicina, sendo 6 provenientes do MS e 18 de SP. Portanto, através do sequenciamento de Sanger, foi possível detectar um número maior de isolados mutados e mais mutações quando comparado ao teste GenoType® MTBDRplus. Isso acontece porque na técnica de sequenciamento, um fragmento do gene como um todo é analisado e no caso do teste GenoType® MTBDRplus, é verificada apenas a ausência ou presença das mutações mais frequentes descritas na literatura, além de não ser analisado o gene ahpC. A grande ...

Estudo dos fatores de virulência e resistência à oxacilina em Staphylococcus spp. isolados de aspirado traqueal de pacientes em ventilação mecânica internados em UTI

Patients that are mechanically ventilated in ICUs are constantly exposed to different pathogens, which present multiantibiotic resistance. Among these microorganisms, is MRSA (Meticillin-Resistant Staphylococcus aureus) considered to be a therapeutic challenge due to its resistance to β-lactam antibiotics. Therefore, this study proposed to identify species of Staphylococcus spp. isolated from mechanically ventilated patients in ICU, the gene mecA detection and the genes of the enterotoxins A (sea), B (seb), C (sec-1) and D (sed) in samples of S. aureus, as well as the phenotypic resistance determination to oxacillin using the disc-diffusion method with discs of oxacillin and cefoxitin. The samples collection occurred during in a period of 19 months, obtaining samples from 232 patients. A percentage of 39% (70) of Gram-positive cocci were found; which 82,8% (58) were identified as Staphylococcus spp,. among these, 75,8% (44) corresponded to S. aureus species and 47,7% were identified as MRSA. It was found resistance to both drugs in 31,8% of the S. aureus samples, 16 (36,3%) had the gene sea and 11 (25%) had the sec-1 gene. Among the coagulase-negative staphylococci obtained, the species most found was S. epidermidis, corresponding to 43% (6). The results revealed that one of the most important etiologic agents of VAP amid the Gram-positive cocci is the species S. aureus, with special attention to MRSA. The presence of enterotoxins genes in S. aureus did not showed determinant role in VAP, but the presence of these superantigens can contribute worsening the patient’s prognosis, since they are associated with intense inflammatory response

Indirect cytocompatibility of a low-concentrated hydrogen peroxide bleaching gel to odontoblast-like cells

Aim To assess the initial cytotoxicity and the late phenotype marker expression of odontoblast-like cells (MDPC-23) subjected to less aggressive in-office bleaching therapies. Methodology A 17.5% hydrogen peroxide (H2O2) gel was applied for 45, 15 or 5 min to enamel/dentine discs adapted to trans-wells positioned over cultured MDPC-23 cells. No treatment was performed on the negative control. Immediately after bleaching, the cell viability, gene expression of inflammatory mediators and quantification of H2O2 diffusion were evaluated. The ALP activity, DSPP and DMP-1 gene expression and mineralized nodule deposition (MND) were assessed at 7, 14 or 21 days post-bleaching and analysed statistically with Mann–Whitney U-tests (α = 5%). Results H2O2 diffusion, proportional to treatment time, was observed in all bleached groups. Reductions of approximately 31%, 21% and 13% in cell viability were observed for the 45-, 15- and 5-min groups, respectively. This reduction was significant (P < 0.05) for the 45- and 15-min groups, which also presented significant (P < 0.05) over-expression of inflammatory mediators. The 45-min group was associated with significant (P < 0.05) reductions in DMP-1/DSPP expression at all periods, relative to control. The ALP activity and MND were reduced only in initial periods. The 15-min group had less intense reduction of all markers, with no difference to control at 21 days. Conclusions The 17.5% H2O2 applied to tooth specimens for 5 min caused no alteration in the odontoblast-like cells. When this gel was applied for 45 or 15 min, a slight cytotoxicity, associated with alterations in phenotypic markers, was observed. However, cells were able to recover their functions up to 21 days post-bleaching.

Mutações putativo-causais em genes candidatos associadas à fertilidade de bovinos de corte e bubalinos

Pós-graduação em Genética e Melhoramento Animal - FCAV

FIPRONIL promotes motor and behavioral changes in honey bees (APIS Mellifera) and affects the development of colonies exposed to sublethal doses

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Carotenoid Profile of Tomato Sauces: Effect of Cooking Time and Content of Extra Virgin Olive Oil

The consumption of carotenoid-rich vegetables such as tomatoes and tomato sauces is associated with reduced risk of several chronic diseases. The predominant carotenoids in tomato products are in the (all-E) configuration, but (Z) isomers can be formed during thermal processing. The effect of cooking time (15, 30, 45 and 60 min) and the addition of extra virgin olive oil (5% and 10%) on the carotenoid extractability of tomato sauces was monitored using liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) and LC-ultraviolet detection (LC-UV). The thermal treatment and the addition of extra virgin olive oil increased the levels of antioxidant activity, total carotenoids, Z-lycopene isomers, -carotene and -carotene. These results are of particular nutritional benefit since higher lycopene intake has been associated with a reduced risk of lethal prostate and a reduction of prostate-specific antigen (PSA) levels. Moreover, -carotene has been reported to suppress the up-regulation of heme oxygenase-1 gene expression in a dose dependent manner and to suppress UVA-induced HO-1 gene expression in cultured FEK4.

Genome-wide scan for visceral leishmaniasis in mixed-breed dogs identifies candidate genes involved in T helper cells and macrophage signaling

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito Diabetes mellitus gestacional na modulação de genes relacionados ao metabolismo mitocondrial e a implicação na predisposiçao do recém-nascido à obesidade

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estratégias para minimização do estresse oxidativo em sistemas de produção in vitro de embriões bovinos destinados à vitrificação

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Diabetes e o polimorfismo Arg972: atualização bibliográfica

The objective of this update was to perform an evaluation of articles published in recent years about the Arg972 polymorphism of IRS-1 gene and its role in diabetes. It was used the database site of National Center of Biotechnology Information (NCBI - PUB- -MED). For search of articles the terms “polymorphisms on IRS-1 and diabetes” were used. Approximately 54% of the articles found were related to the polymorphism of interest and of these, approximately 68% the polymorphism was linked to various forms of diabetes. The Arg972 polymorphism is associated with decreased insulin synthesis and secretion with reduced stimulation of the activity of insulin pathway PI3K, MAPK and thymidine incorporation. It more frequently in patients with type 2 diabetes and gestational diabetes, showing a direct connection with the status of obesity and insulin resistance.

Comparação de métodos para extração de dna na reação em cadeia da polimerase para detecção de Salmonella enterica sorovar enteritidis em produtos avícolas

The identification of Salmonella spp. in food samples by microbiological diagnosis is time consuming, with approximately five different stages, requiring about 120 hours until the final result. The utilization of the polymerase chain reaction technique (PCR) can reduce this time, but substances present in samples may affect the reaction. The present work aimed to compare DNA extraction by thermic treatment and by the use of cetyltrimethil ammonium bromide (CTAB), in products originated from poultry houses corresponding to raw material (meat meal) and experimentally contaminated drag swabs. Materials obtained from the extractions were submitted to PCR, utilizing a pair of initiator oligonucleotides for amplification of Sdf 1 gene fragments. Comparing the methods of extraction, it was observed that when CTAB was employed, SE was detected in 70% of meat meal and in 80% of drag swabs, while the thermic treatment method yielded positive results in 20% of meat meal and in 40% of drag swabs. SE was detected under both methods utilized for DNA extraction, but the use of CTAB detected a greater number of positive samples, compared with thermal treatment.

Intensidade do estresse hídrico modula a expressão de aquaporinas durante a reidratação em Sorghum bicolor

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Differential Expression of Genes Involved in the Degeneration and Regeneration Pathways in Mouse Models for Muscular Dystrophies

The genetically determined muscular dystrophies are caused by mutations in genes coding for muscle proteins. Differences in the phenotypes are mainly the age of onset and velocity of progression. Muscle weakness is the consequence of myofiber degeneration due to an imbalance between successive cycles of degeneration/regeneration. While muscle fibers are lost, a replacement of the degraded muscle fibers by adipose and connective tissues occurs. Major investigation points are to elicit the involved pathophysiological mechanisms to elucidate how each mutation can lead to a specific degenerative process and how the regeneration is stimulated in each case. To answer these questions, we used four mouse models with different mutations causing muscular dystrophies, Dmd (mdx) , SJL/J, Large (myd) and Lama2 (dy2J) /J, and compared the histological changes of regeneration and fibrosis to the expression of genes involved in those processes. For regeneration, the MyoD, Myf5 and myogenin genes related to the proliferation and differentiation of satellite cells were studied, while for degeneration, the TGF-beta 1 and Pro-collagen 1 alpha 2 genes, involved in the fibrotic cascade, were analyzed. The result suggests that TGF-beta 1 gene is activated in the dystrophic process in all the stages of degeneration, while the activation of the expression of the pro-collagen gene possibly occurs in mildest stages of this process. We also observed that each pathophysiological mechanism acted differently in the activation of regeneration, with distinctions in the induction of proliferation of satellite cells, but with no alterations in stimulation to differentiation. Dysfunction of satellite cells can, therefore, be an important additional mechanism of pathogenesis in the dystrophic muscle.

High prevalence of OXA-143 and alteration of outer membrane proteins in carbapenem-resistant Acinetobacter spp. isolates in Brazil

Carbapenem resistance amongst Acinetobacter spp. has been increasing in the last decade. This study evaluated the outer membrane protein (OMP) profile and production of carbapenemases in 50 carbapenem-resistant Acinetobacter spp. isolates from bloodstream infections. Isolates were identified by API20NE. Minimum inhibitory concentrations (MICs) for carbapenems were determined by broth microdilution. Carbapenemases were studied by phenotypic tests, detection of their encoding gene by polymerase chain reaction (PCR) amplification, and imipenem hydrolysis. Nucleotide sequencing confirming the enzyme gene type was performed using MegaBACE 1000. The presence of OMPs was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and PCR. Molecular typing was performed using pulsed-field gel electrophoresis (PFGE). All isolates were resistant to carbapenems. Moreover, 98% of the isolates were positive for the gene encoding the enzyme OXA-51-like, 18% were positive for OXA-23-like (only one isolate did not show the presence of the insertion sequence ISAba1 adjacent to this gene) and 76% were positive for OXA-143 enzyme. Five isolates (10%) showed the presence of the IMP-1 gene. Imipenem hydrolysing activity was detected in only three strains containing carbapenemase genes, comprising two isolates containing the bla(IMP) gene and one containing the bla(OXA-51/OXA-23-like) gene. The OMP of 43 kDa was altered in 17 of 25 strains studied, and this alteration was associated with a high meropenem MIC (256 mu g/mL) in 5 of 7 strains without 43 kDa OMP. On the other hand, decreased OMP 33-36 kDa was found in five strains. The high prevalence of OXA-143 and alteration of OMPs might have been associated with a high level of carbapenem resistance. (C) 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

GREB1 tissue expression is associated with organ-confined prostate cancer

Objective: By reason of its heterogeneous behavior, it is difficult to determine the prognosis of many prostate cancer cases. Patients with the same clinicopathologic conditions may present varying clinical findings and rates of progression. We determined the role of new genes as potential molecular markers for prostate cancer prognosis. Materials and methods: We performed a microarray analysis of two pools of patients with prostate cancer divided according to their clinicopathologic characteristics. After that, we validated these results by testing the genes with most different expressions between the two pools using the quantitative real time polymerase chain reaction method. We analyzed gene expression in 33 patients with localized prostate cancer according to prostate specific antigen (PSA), pathologic stage, Gleason score, and biochemical recurrence. For statistical analysis we used the Mann-Whitney Test. Results: The microarray analysis revealed that 4,147 genes presented a different expression between the two pools. Among them, 3 genes, TMEFF2, GREB1, and THIL,, were at least 13-times overexpressed, and 1 gene, IGH3, which was at least 5times under-expressed in pool 1 (good prognosis) compared with pool 2 (bad prognosis), were selected for analysis. After the validation tests, GREB1 was significantly more overexpressed among patients with stage T2 compared with T3 (P = 0.020). The expressions of other 3 genes did not present significant differences according to the clinicopatholoOcal variables. Conclusions: Tissue expression of GREB1 is associated with organ-confined prostate cancer and may constitute a gene associated with a favorable prognosis. (C) 2012 Elsevier Inc. All rights reserved.